1. Field of the Invention
This invention relates to a strain E.coli JM83/pKP2 transformed with a novel plasmid and phytase produced therefrom and, more particularly, to a strain E.coli JM83/pKP2 transformed with a novel recombinant vector pKP1 or pKP2, so prepared by a gene manipulation, through elucidating the gene sequence intended for the mass production of novel phytase serving the role to enhance the phosphorous bioavailability in grains used as livestock feeds.
2. Description of the Prior Art
Phytase is an enzyme which degrades phytic acid into phosphate and phosphate inositol. 50.about.70% of phosphate in grain used as livestock feeds exists in the form of phytic acid, but phytase is not present in monogastric animals such as hens and hogs, thus resulting in low phosphate availability. Further, indigested phytic acid phytate released to a water source has become one of the serious environment contamination sources and causes eutrophication in small lakes or tides. Further, monogastric animals can not utilize phytic acid in their intestine due to its chelation with a trace amount of minerals, amino acids and vitamins which are essential for the metabolism of livestock. These formed water insoluble and indigestible chelate-complexes released in the form of feces are responsible for the change of the environmental ecosystem, thus inducing a serious environmental contamination.
In view of these situations, the application of phytase into the livestock feeds can reduce the supply of inorganic phosphate due to an increase of phosphate bioavailability in livestock, thus leading to economic benefits. In addition, the improved availability of phosphate and other bioactive substances may also contribute much to the reduction of the environmental contamination.
In particular, the utilization of phytase in livestock is very important in that the law regulating the amount of phosphate in animal waste was established in 1996 in Korea and, in addition to that, it has been mandatory to add phytase in the feeds of animals in the European countries. Further, when phytase i added to the feeds, it may greatly improve the productivity of livestock by enhancing the availability of some bioactive substances such as vitamins and amino acids, including some trace elements such as calcium and zinc ions whose activity is reduced by chelation with negatively charged phytate. As such, the use of feeds containing phytase in livestock can enhance the availability of feeds and reduce the environmental contamination cause by phosphate.
From the aforementioned benefits, the intensive studies with respect to phytase including the effects of phytase on animals (L. G. Yound et al., 1993; X. G. Lei et al., 1994; Z. Morez et al., 1994) have been performed mainly in Europe (A. H. J. Ullah et al., 1994; K. C. Ehrich, 1994; C. S. Piddington, 1993). However, since phytase can cleave a limited number of phosphate only and is mostly produced by molds which have slow growth rate, it is not economical in terms of mass production. In addition, it is difficult to use the phytic acid as an additive for monogastric animals since it is undesirable for their physiological characteristics.
The inventor, et al. have performed intensive studies for overcoming the above problems associated with phytase. As a result, a novel strain Bacillus sp. DS-11 producing phytase with an excellent activity and different characteristics over the conventional phytase was identified and deposited to the Korean Collection for Type Cultures within the Korea Research Institute of Bioscience and Biotechnology affiliated with Korea Institute of Science and Technology (KCTC 0231BP), the Korean Patent Strain Depository Institute. The above patent application was filed with the Korean Industrial Patent Office (The Korean Patent Appl. No.: 96-6817). Hence, various characteristics on a novel phytase produced from the microorganism were investigated and, as a result, the novel phytase proved to be excellent on heat and pH with better stability.
From the above results, the inventor et al. sequenced the DNA by cloning some phytase-coding gene in a strain Bacillus sp. DS-11 under the patent application so as to ensure the mass production of a novel phytase having the above excellent characteristics. As a result, the phytase-coding gene sequence Bacillus sp. DS-11 was recognized to be a novel one, being entirely different from that of Aspergillus awamori(WO 94-3072A), Aspergillus ficum (EP 42038, U.S. Pat. No. 5,436,156), Aspergillus niger(EP 420358) and Aspergillus terreus(EP 684313) among the genes clones hitherto. Thus, its accessory No. U85968(date Jan. 21, 1997) was given from GenBank of NCBI in the U.S.A.
Next, the inventor et al. transformed E.coli with the plasmid vector (pKP1 or pKP2 ) encoding the phytase gene of Bacillus sp. DS-11, and the transformed strain E.coli JM83/pKP2 was deposited at the Korean Collection Type Cultures within the Korea Research Institute of Bioscience and Biotechnology affiliated with the Korea Institute of Science and Technology (KCTC 0308BP dated Jan. 28, 1997), the Korean Patent Strain Depository Institute.